Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production

Abstract

Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

Production of Value-Added Chemicals by Bacillus methanolicus Strains Cultivated on Mannitol and Extracts of Seaweed Saccharina latissima at 50°C

“…The recombinant cell culture reached a cell dry weight of about 55% compared to the biomass typically obtained under analogous methanol fermentations ( Table 5). The highest cadaverine production titer achieved was 6.3 g/l (concentration corrected for dilution due to the feeding) corresponding to ∼60% of the previously reported production level obtained during fedbatch methanol fermentation (Irla et al, 2016a). Interestingly, the calculated production yield per cell dry weight was higher on mannitol (0.19 g/g CDW) than on methanol (0.17 g/g CDW).…”

Methanol as carbon substrate in the bio‐economy: Metabolic engineering of aerobic methylotrophic bacteria for production of value‐added chemicals

“…The expression of cadA in B. methanolicus MGA3 led to the highest production of 11.3 g/L in a fed‐batch methanol fermentation . Cadaverine production titers could be increased to 17.5 g/L using a theta‐replicating expression vector instead of a rolling‐circle‐replicating vector, which is even higher than the best l‐ lysine titers obtained with this organism (see above) . These represented the first examples of cadaverine production from methanol.…”

“…Individual overexpression of these genes in B. methanolicus MGA3 increased l‐glutamate production yields from 0.8 g/L to 1.1 g/L under small‐scale methanol cultivations, however, the recombinant strains displayed no l‐ glutamate overproduction when tested in fed‐batch methanol fermentations. The reason for the latter is likely due to plasmid loss under such growth conditions, which now likely can be circumvented by using the new and better genetic tools applicable for this organism . Another example is Methylobacillus glycogenes with isolated strains secreting more than 30 g/L of l‐ glutamate .…”

Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences

“…Taken together with the results for pBAS2, these data suggest that recA is required for stable pBAS2 replication specifically, and that replication, not DNA entry into the cell, is affected by the deletion. Nearly all plasmids so far described for thermophilic Gram-positive bacteria replicate by a rolling-circle mechanism [41, 45], although the moderate thermophile Bacillus methanolicus was recently shown to maintain a theta-replicating plasmid at a lower temperature [23]. It is noteworthy that ssDNA intermediates are usually formed during rolling-circle replication, and that RecA protein may be required for protection of the DNA [44] or for replication restart mechanisms [32] necessary for faithful replication of the plasmid.…”